Chimeric non-human animals comprising human hepatocytes

Abstract

The instant disclosure relates to genetically modified non-human animals comprising deletions of the Il2rg gene, the Fah gene and the Prkdc gene, as well as methods of making and methods of using the same.

Description

CHIMERIC NON-HUMAN ANIMALS COMPRISING HUMAN HEPATOCYTESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 564, 307#filed March 12, 2024, which is incorporated herein by reference in its entirety.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into fee specification. The name of fee XML file containing fee Sequence Listing XML is “ A VCR-004-00 lWO_SeqList. xml”. The XML file is 67,276 bytes, created on March 10, 2025, and is being submitted electronically via USPTO Patent Center.FIELD

[0003] The instant disclosure relates to genetically modified non-human animals comprising deletions of the I12rg gene, the Fab gene and fee Prkdc gene, as well as methods of making and methods of using fee same.BACKGROUND:

[0004] The Prkdc-SCID mutation occurred spontaneously in an inbred mouse colony, C.B- 17 mice, maintained at the Fox Chase in Philadelphia, PA, USA(Bosma, et al. Nature 301, 527- 530 (1983)). The severe combined immune deficiency (SCID) spontaneous mutation Prkdc Prkdc, coding for the DNA-dependent protein kinase catalytic subunit, results in an absence of functional T cells and B cells, lymphopenia, hypogammaglobulinemia, and a normal hematopoietic microenvironment (Bosma, et al. Nature 301, 527-530 (1983)). Prkdc-SCID mice are commonly used for human hematopoietic cell engrafhnent (Shultz, et al, Nature reviews 7, 118-130 (2007)).

[0005] The existing chimeric animal models containing human hepatocytes have several limitations, including low human liver chimerism, interference of mouse tissue for evaluation of human liver parameters, opportunistic infections, reduced life and health span, low fertility and other husbandry issues. Also, chimeric animal models are often used when older after full humanization and fee tail veins are difficult to identify in non-albino (tyrosinase deficient mice) animals. Therefore, there remains an unmet need for new ami improved genetically modified animals with human hepatocytes.SUMMARY[00061 In one aspect, provided herein is a genetically modified non-human animal, comprising a deletion of the H2rg gene, the Fah gene and the Prkdc gene. In some embodiments, a genetically modified non-human animal further comprises a deletion of the aavr gene, hi some embodiments, a genetically modified non-human animal further comprises a deletion of the por gene. In some embodiments, a genetically modified non- human animal further comprises a deletion of the tyrosinase gene. In some embodiments, the non-human animal is a primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse, hi some embodiments, the non-human animal is a rat. In some embodiments, at least 75% of the hepatocytes present in the non- human animal are human hepatocytes. In some embodiments, at least 80% of the hepatocytes present in the non-human animal are human hepatocytes. In some embodiments, at least 85% of tiie hepatocytes present in the non-human animal are human hepatocytes. hi some embodiments, the non-human animal lacks autogenous hepatocytes.

[0007] In some embodiments, the non-human animal comprises a wildtype rag2 gene.

[0008] In some embodiments, the non-human animal is immunodeficient.

[0009] In another aspect, provided herein is a method of evaluating hepatotoxicity of a test substance, the method comprising (a) administering a test substance to the genetically modified non-human animal described herein; (b) measuring at least one indicator of toxicity in the genetically modified non-human animal to which the test substance is administered; and (c) evaluating the effect of the test substance on human hepatocytes by comparing the indicator of toxicity measured in the genetically modified non-human animal to the same indicator of toxicity measured in a genetically modified non-human animal to which no test substance is administered. In some embodiments, the test substance is a medicament intended for human use. In some embodiments, the at least one indicator of toxicity is blood human albumin, body weight, hver-weight-to-body-weight ratio, total blood albumin, total blood protein, blood alanine aminotransferase (ALT), blood aspartate aminotransferase (AST), Gamma-Glutamyl Transferase (GGT), alkaline phosphatase (ALP) or total blood bilirubin

[0010] In another aspect, provided herein is a method for preparing a genetically modified non-human animal comprising human hepatocytes, comprising steps of: (a) providing a non- human animal comprising a deletion of the Il2rg gene, the Fah gene and the Prkdc gene; and (b) transplanting human hepatocytes into the non-human animal. In some embodiments, the genetically modified non-human animal further comprises a deletion of the aavr gene and / orthe por gene. In some embodiments, a genetically modified non-human animal further comprises a deletion of the tyrosinase gene. In some embodiments, the non-human animal is a primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse or rat. hi some embodiments, at least 75% of the hepatocytes present in the non-human animal are human hepatocytes, hi some embodiments, at least 80% of the hepatocytes present in the non-human animal are human hepatocytes. In some embodiments, at least 85% of the hepatocytes present in the non-human animal are human hepatocytes. In some embodiments, the non-human animal lacks autogenous hepatocytes. In some embodiments, the non-human animal comprises a wildtype rag2 gene. In some embodiments, the non-human animal is immunodeficient.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows human specific albumin measurements in humanized and not humanized (control) PIES (Por fl / fl, I12rg- / -,Fah- / -, Prkdc-SCID + / + and wildtype for the Rag2 (Rag2+ / +)) mice expressed in mg / L human albumin in the murine serum.

[0012] FIG. 2 shows engrafiment of three independent donor hepatocytes into IRS (I12rg- / - ZFah- / - / Prkdc-SCID + / +) mice. Each dot represents one transplanted and evaluated mouse sample (N=4 for donor A and B and N=7 for donor C).

[0013] FIG. 3 shows a comparison human chimerism of IFS (I12rg- / - / Fah- / - / Prkdc-SCID + / +) mice to Fah- / - / Rag2- / - / I12rg- / - (known as FRG or URF) mice assessed by human albumin levels in the murine plasma. IFS (N=8) and seven URF (N=7) mice. Statistical analysis is done by PRISM Version 10.4.1. using Mann-Whitney test (two-tailed), p value (*) < 0.05.

[0014] FIG. 4 shows immunostaining of chimeric liver of human liver chimerism of IFS (I12rg- / - / Fah- / - / Prkdc-SCID + / +) mice. N=8; representative picture is shown with dark areas reactive to human lactate dehydrogenase (hLDH).

[0015] FIG. 5 shows immunostaining of chimeric liver of human liver chimerism of PIFS (Por fl / fl, I12rg- / -,Fah- / -, Prkdc-SCID + / + ) mice with deleted P450 oxidoreductase (Por) gene by adenoviral vector (CRE-Ad). Representative pictures of chimeric liver stained with hematoxylin and eosin (H&E; FIG. 5 A), human specific lactate dehydrogenase (hi UH; FIG. 5B) and fumarylacetoacetate hydrolase (FAH; FIG 5C) are shown.

[0016] FIG. 6 shows immunostaining of chimeric liver of human liver chimerism of IFS A (I12rg- / -,Fah- / -, Prkdc-SCID + / + AAVRV") mice. Representative picture of chimeric liver isshown stained with hematoxylin and eosin (H&E) (left), and immunostaining for human specific lactate dehydrogenase (hLDH) (right).

[0017] FIG. 7 shows immunostaining of highly humanized (>50%) chimeric liver of human liver chimerism of IFS (I12rg- / -,Fah- / -, Prkdc-SCID + / + ) mice. Representative serial section from a lobe of a high humanization chimeric liver is shown stained with hematoxylin and eosin (H&E) (top), and immunostaining for human specific lactate dehydrogenase (hLDH) (middle) and fumarylacetoacetate hydrolase (FAH) (bottom) .

[0018] FIG. 8 shows a comparison human chimerism of IFSA (I12rg- / - / Fah- / - / Prkdc-SCID + / + AAVR? ) mice to Fah- / -ZRag2- / - / I12rg- / - AAVR7) (11KFA) mice assessed by human albumin levels in the murine plasma; n=8; Statistical analysis is done by PRISM Version 10.4.1. using Mann-Whitney test (two-tailed), p value (*) < 0.05.DETAILED DESCRIPTION

[0019] The present disclosure provides a chimeric non-human animal comprising human hepatocytes, hi particular, the present disclosure relates to non-human animals modified to comprise deletions in the interleukin 2 receptor gamma chain (fl2rg) gene, the fumarylacetoacetate hydrolase (Fah) gene and / or the DNA-dependent protein kinase catalytic subunit (Prkdc) gene and which comprise human hepatocytes. The present disclosure also provides methods of making and using the genetically modified non-human animals.Genetically Modified Animals

[0020] In one aspect, provided herein is a genetically modified non-human animal, comprising a deletion of the H2rg gene, the Fah gene and the Prkdc gene. These animals may be repopulated with human liver cells. Generally, the non-human animals are immunocompromised to allow for the transplantation of human liver cells.

[0021] In some embodiments, a genetically modified non-human animal disclosed herein further comprises a deletion of the aavr gene. In some embodiments, a genetically modified non-human animal disclosed herein further comprises a deletion of the por gene. In some embodiments, the genetically modified non-human animal disclosed herein further comprises a deletion of the tyrosinase gene. In some embodiments, a genetically modified non-human animal disclosed herein further comprises a deletion of the aavr gene and of the por gene. In some embodiments, a genetically modified non-human animal disclosed herein furflier comprises a deletion of the aavr gene and the tyrosinase gene. In some embodiments, a genetically modified non-human animal disclosed herein further comprises a deletion of the,the por gene and the tyrosinase gene. In some embodiments, a genetically modified nonhuman animal disclosed herein further comprises a deletion of the aavr gene, die por gene and the tyrosinase gene.

[0022] A genetically modified non-human animal comprising deletions of il2rg,fah and prkdc is also referred to as an ’TFS” animal. An genetically modified non-human animal comprising deletions of il2rg, fah, prkdc and por is also referred to as a “PIES” animal. A genetically modified non-human animal comprising deletions of il2rg,fah, prkdc and aavr is also referred to as an “IPS A” animal. An animal comprising deletions of iterg^fah, prkdc, por and aavr is also referred to as a “PIFSA” animal.

[0023] In some embodiments, the non-human animal does not comprise a mutation in the rag2 gene. Thus, in some embodiments, the non-human animal expresses wildtype Rag2 protein.

[0024] The non-human animals described herein may be transplanted with human hepatocytes using any suitable method known in the art or described herein. Generally, it is desirable to achieve as many human liver cells in the liver of the non-human animal as possible. Human albumin may be used as an indicator of human liver cells and can be used to calculate the degree of chimerism (i.e., the proportion of human liver cells in the liver of the non-human animal; see Bissig et al., (2010)) The Journal of clinical investigation 120, 924- 930). The levels of human hepatocytes in the liver of a non-human animal described herein may be determined at any appropriate time point, for example, 2 months, 3 months, 4 months, 5 months or 6 months after transplantation.

[0025] In some embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of all the hepatocytes in the liver of the non-human animal are human hepatocytes. In some embodiments, about 20 to about 30%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% about 90% to about at least 95% all the hepatocytes in the liver of the non- human animal are human hepatocytes. In some embodiments, at least about 60% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 3 months after transplantation. In some embodiments, at least about 70% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 3 months after transplantation. In some embodiments, at least about 80% of all the hepatocytes in the liver of tire non-human animal are human hepatocytes 3 months after transplantation. In some embodiments, at least about90% of all die hepatocytes in the liver of the non-human animal are human hepatocytes 3 months after transplantation. In some embodiments, at least about 95% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 3 months after transplantation. In some embodiments, at least about 60% of all the hepatocytes in die liver of the non-human animal are human hepatocytes 4 months after transplantation. In some embodiments, at least about 70% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 4 months after transplantation. In some embodiments, at least about 80% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 4 months after transplantation. In some embodiments, at least about 90% of all the hepatocytes in the liver of the non-human animal are human hepatocytes 4 months after transplantation. In some embodiments, at least about 95% of all the hepatocytes in the liver of tire non-lmman animal are human hepatocytes 4 months after transplantation.

[0026] In some embodiments, the non-lmman animal is a primate, bird, mouse, rat, fowl, dog, cm, cow, horse, goat, camel, sheep or pig. In some embodiments, the non-human animal is a rodent, hi some embodiments, the non-human animal is a mouse, n some embodiments, the non-human animal is a rat.Methods of Producing

[0027] The present disclosure also provides a method for preparing a genetically modified non-human animal comprising human hepatocytes.

[0028] In some embodiments, the method comprises: (a) providing a non-human animal comprising a deletion of the Il2rg gene, the Fah gene and die Prkdc gene; and (b) transplanting human hepatocytes into the non-human animal.

[0029] A selection pressure may be applied to increase the liver chimerism in the transgenic non-human animal. Selection pressure for die transplanted human hepatocytes may be generated by, for example, generating a selection pressure against the non-human hepatocytes in the same liver. The genetically modified non-human animals disclosed herein are deficient in Fall and tiierefore accumulates toxic catabolites in the non-human hepatocytes, which eventually leads to apoptosis. However, if the drug nitisinone (NTBC) is supplied, these toxic catabolites do not accumulate in die non-human, Fah-deficient animal due to substrate reduction. In contrast, the human hepatocytes transplanted into the non-human animal have a functional Fah enzyme and therefore do not accumulate toxic catabolites and do not undergo apoptosis, even without the drug nitisinone (NTBC). Therefore, supplying and subsequentlywithdrawing NTBC may increase the number of human hepatocytes present in the liver of a genetically modified non-human animal described herein.

[0030] In some embodiments of tire methods of this disclosure, applying a selection pressure comprises not providing nitisinone (NTBC) to the non-human animal of step (b). Thus, in some embodiments of tire methods of this disclosure, the method further comprises auuplying NTBC (e.g., in the drinking water) followed by removal of NTBC following step (b) of the methods disclosed herein.

[0031] In some embodiments of the methods of this disclosure, the genetically modified nonhuman animal does not comprise a transgene. In some embodiments, the genetically modified non-human animal further comprises a transgene. In some embodiments of the methods of this disclosure, the transgene is an antibiotic resistance cassette.

[0032] In some embodiments, the method of making a genetically modified non-human animal disclosed herein utilizes a conditional (CRE / LoxP system) deleted Por gene (e.g., to generate PIFS and PIFSA animals). Animals may be injected intravenously with an adenoviral vector encoding a CRE recombinase under the CMV promoter. One example of such a vector is tire Ad5 CMV-Cre, which may be administered at a dose of 2.3xl0npfu / ml. The por gene may be deleted by administering file vector encoding CRE recombinase at any suitable time point, for example, 24 hours before hepatocyte transplantation. Alternatively or additionally, the vector may be administered when the liver in the non-human animal reaches a high level of human chimerism (e.g., greater than 70%).

[0033] A person of skill in the art will appreciate that a gene may be deleted or mutated using any suitable method know in the art. Several methods of genetically modifying non-human animals are known in file art. For example, an animal may be rendered defective in the II2rg gene, the Fah gene, the Prkdc gene, the por gene and / or the aavr gene using CRISR-Cas9 technology or by knockdown or knockout of the target gene. Knockdown of a gene may be achieved using, for example, exogenous agents, RNA interference technology7, or floxed allele of the target gene. Some mutations occur spontaneously, such as the SCID mutations in prkdc, resulting in DNA-PK deficiency.

[0034] A human nucleic sequence encoding an exemplary II2-rg protein of the disclosure consist of or comprises Genbank Accession number: NM 000206.3 (SEQ ID NO: 1). The corresponding human amino acid sequence of an exemplary II2-rg protein of tire disclosure consist of or comprises Genbank Accession number: NP 000197.1 (SEQ ID NO: 2)

[0035] A murine nucleic sequence encoding an exemplary II2-rg protein of the disclosure consist of or comprises Genbank Accession number: NM 013563.4 (SEQ ID NO: 3). Thecorresponding murine amino acid sequence of an exemplary II2-rg protein of the disclosure consist of or comprises Genbank Accession number: NP 03859L1 (SEQ ID NO: 4).

[0036] A human nucleic sequence encoding an exemplary Fah protein of the disclosure consist of or comprises Genbank Accession number: NM 000137.4 (SEQ ID NO: 5). The conesponding human amino acid sequence of an exemplary Fah protein of the disclosure consist of or comprises Genbank Accession number: NP 000128.1 (SEQ ID NO: 6).

[0037] A murine nucleic acid sequence encoding an exemplary Fah protein of the disclosure consist of or comprises Genbank Accession number: NM 010176.4 (SEQ ID NO: 7). The corresponding murine amino add sequence encoding an exemplary Fah protein of the disclosure consist of or comprises Genbank Accession number: NP 034306.2 (SEQ ID NO: 8).

[0038] The adeno-associated virus (AAV) receptor (AAVR) disclosed herein, also referred to as Dyslexia-associated protein KIAA0319-like (KIAA0319L) protein, is a predicted type I transmembrane protein with five Ig-like domains in its ectodomain, referred to as polycystic kidney disease (PKD) domains. Ig-like domains mediate cell-cell adhesion and are present in various well-characterized virus receptors, including those for poliovirus, measles virus and reovirus. (Pillay S. et al., Nature 2016;530: 108-112) The PKD of AAVR have been shown to bind directly to tire spike region of the AAV2 capsid adjacent to the icosahedral three-fold axis. (Zhang R. et al., Nat Microbiol, 2019 Apr;4(4):675-682).

[0039] A human nucleic sequence encoding an exemplary AAVR protein of the disclosure consist of or comprises Genbank Accession number NM 024874.5 (SEQ ID NO: 9). The corresponding human amino acid sequence of an exemplary AAVR protein of the disclosure consist of or comprises Genbank Accession number: NP 079150.3 (SEQ ID NO: 10).

[0040] A murine nucleic sequence encoding an exemplary AAVR of the disclosure consist of or comprises Genbank Accession number NM 001035526.1 (SEQ ID NO: 11). The corresponding murine amino acid sequence of an exemplary AAVR of the disclosure consist of or comprises Genbank Accession number: NP 001030603.1 (SEQ ID NO: 12).

[0041] A human nucleic sequence encoding an exemplary DNA-PK protein of the disclosure consist of or comprises Genbank Accession number: NM 001081640.2 (SEQ ID NO: 13). The conesponding human amino acid sequence encoding an exemplary DNA-PK protein of the disclosure consist of or comprises Genbank Accession number: NP 001075109.1 (SEQ ID NO: 14).

[0042] A murine nucleic sequence encoding an exemplary DNA-PK protein of the disclosure consist of or comprises Genbank Accession number: NM 011159.2 (SEQ ID NO: 15). Thecorresponding murine amino acid sequence encoding an exemplary DNA-PK protein of the disclosure consist of or comprises Genbank Accession number: NP 035289.2 (SEQ ID NO: 16)-

[0043] A known mutation that results in DNA-PK deficiency is fire SCID mutation. This mutation introduces a premature stop codon, resulting in non-fimctional DNA-PK protein.

[0044] Human p450 cytochrome clusters contain 57 putatively functional genes and 58 pseudogenes, while the mouse cytochrome clusters are greatly expanded accounting for 102 putatively functional genes and 88 pseudogenes. Several mutations in Por have been described.

[0045] Animals carrying the described combinations of mutations may then be generated by crossing animal strains. For example, an IF A animal may be generated by crossing an IFS animal with an IFSA animal. PIFS animals may be generated by crossing a PIF animal with a Prkdc-SCID animal, a PIFSA animal may be generated by crossing an IFSA animal with a PIES animal. A person of skill in the art will be able to confirm that an animal carries the desired genotype using widely available genotyping platforms.

[0046] The human hepatocytes to be used for transplantation can be human hepatocytes isolated from normal human liver tissue by any suitable method known in the art or described herein, such as a collagenase perfusion. The thus separated hepatocytes can also be used after cryopreservation.

[0047] Alternatively, hepatocytes can be separated by a technique such as a collagenase perfusion method from a chimeric mouse liver, in which mouse hepatocytes have been replaced by human hepatocytes, can be used in a fresh state, and the cryopreserved chimeric mouse hepatocytes are also available after thawing.

[0048] Human hepatocytes can be transplanted into the genetically modified non-human animal via any suitable route. For example, human hepatocytes may be transplanted into the spleen of a genetically modified non-human animal of the present disclosure. Alternatively, human hepatocytes can be transplanted via the portal vein of the genetically modified non- human animal.

[0049] The number of human hepatocytes to be transplanted may range from about 1 to 2,000,000 cells, and preferably range from about 200,000 to 1 ,000,000 cells. In some embodiments, about 250,000 to about 500,000 human hepatocytes are transplanted into an animal. In some embodiments, about 500,000 to about 750,000 human hepatocytes are transplanted into an animal. In some embodiments, about 750,000 to about 1,000,000 human hepatocytes are transplanted into an animal. In some embodiments, about 1,000,000 to about1,500,000 human hepatocytes are transplanted into an animal, hi some embodiIs, about 1,500,000 to about 2,000,000 human hepatocytes are transplanted into an animal.

[0050] The gender of the genetically modified non-human animal of the present disclosure is not particularly limited. Male or female animals may be used to generate the genetically modified animals described herein.

[0051] Similarly, the age on days of the genetically modified non-human animal of the present disclosure upon transplantation is not particularly limited. Generally, when human hepatocytes are transplanted into a younger animal (e.g., early weeks of age), the human hepatocytes can generally proliferate more actively as fee animal grows. Hence, in some embodiments, fee human hepatocytes are transplanted into a genetically modified non-human animal feat is about 0 to 40 days old. In some embodiments, fee human hepatocytes are transplanted into a genetically modified non-human animal that is about 8 to 40 days old. In some embodiments, fee human hepatocytes are transplanted into a genetically modified non- human animal feat is about 8 to 30 days old. In some embodiments, the human hepatocytes are transplanted into a genetically modified non-human animal feat is about 10 to 40 days old.Methods of Use

[0052] Also provided herein are methods of using the genetically modified non-human animals described herein. One example of a method of using the genetically modified non- human animals described herein is for the assessment of metabolism of a therapeutic agent intended for human use. Most regulatory agencies require extensive animal testing of therapeutic agents that intended for administration to human patients, but an animal differs substantially in their liver composition and function from humans. This makes accurate prediction of human drug metabolism in the test animal challenging. Metabolites of a therapeutic agent or other test substance may be active and contribute to liver toxicity or other effects. The genetically modified non-human animals disclosed herein can be used to more accurately model the metabolism of a substance in humans.

[0053] In one aspect, provided herein is a method of evaluating the metabolism of a test substance, e.g. by screening for and identifying metabolites of the test substance. In some embodiments, a method of evaluating the metabolism of a test substance comprises (a) administering a test substance to the genetically modified non-human animal of the present disclosure; (b) measuring the metabolites present in the genetically modified non-human animal.

[0054] Metabolites may be measured in any suitable sample, for example, blood or urine. Metabolites of a test substance may be identified and quantified using any suitable methods known in the art, including, for example, mass spectrometry-based methods such as LC-MS.

[0055] In another aspect, provided herein is a method of evaluating hepatotoxicity of a test substance, the method comprising (a) administering a test substance to a genetically modified non-human animal disclosed herein; and (b) measuring at least one indicator of toxicity in the genetically modified non-human animal to which the test substance is administered. In some embodiments, the method further comprises step (c) evaluating the effect of the test substance on human hepatocytes by comparing the indicator of toxicity measured in the genetically modified non-human animal to the same indicator of toxicity measured in a genetically modified non-human animal to which no test substance is administered.

[0056] Any indicator of liver toxicity that is of interest may be measured. Examples of such indicators include blood human albumin, body weight, liver- weight-to-body-weight ratio, total blood albumin, total blood protein, blood Alanine Aminotransferase (ALT), blood Aspartate Aminotransferase (AST), total blood bilirubin, creatinine, Blood Urea Nitrogen (BUN), troponin, blood cell count, and TSH. A histological examination of tire liver may also be performed. The indicators of liver toxicity may be determined in any suitable sample, for example, in the blood or urine.

[0057] The test substance may be any substance, such as a drug substance, a pharmaceutical composition, an adjuvant, or a food additive or supplement, or another substance intended for use in humans. In some embodiments, the test substance is a medicament intended for human use. In some embodiments, the test substance is a small molecule drug. In some embodiments, the test substance is a biological agent.EXAMPLESExample 1: Generation of Chimeric Mice Comprising Human Hepatocytes

[0058] This example describes the generation and characterization of a transgenic IFS, IPS A, PIES and PIFSA mice comprising human hepatocytes.MethodsMouse Husbandry

[0059] All mice (6-10 months old, humanized or non-humanized) were maintained under a standard 12-h dark / light cycle with water and chow provided ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC)Crossing of mouse strains and generation of the IFS and IFSA.

[0060] Male (or female) IF A (interleukin 2 receptor gamma chain deficient (II2rg- / -), fumarylacetoacetate hydrolase deficient (fah- / -), Adeno-Associate Virus receptor deficient (Aavr- / -)) mice, which were generated as a side product of tire generation of tire TIRFA (transgene free, U2rg- / -, recombination activating gene 2 (Rag2- / -), fah- / -, Adeno-Associate Virus receptor (Aavr- / -)) strain crossing (described in Barzi et al. Nat Common 2024; 15, 1955) were crossed with Prkdc-SCID mice to generate IRS (I12rg- / -,Fah- / -, Prkdc-SCID + / + and wildtype for the Rag2 (Rag2+ / +) mice and IFSA (I12rg- / -,fah- / -, Prkdc-SCID + / +, Aavr - / - and wildtype for the Rag2 (Rag2+ / +) mice.Crossing of mouse strains and generation of the PIES and PIFSA.

[0061] To generate the different mouse strains (IFS, IFSA, PITS and PIFSA), mice were crossed ami genotyped according to published literature:• Prkdc-SCID'. Quadros et al. J Immunol Methods 2017 (Quadros et al. J Immunol Methods 431, 60-62 (2016))• PorfVfl, H2rg- / -,Fah- / -, Aavr - / - (Barzi et al. , Nature communications 15, 1955 (2024); Barzi et at, Nature communications 8, 39 (2017).

[0062] Male or female PIF (P450 oxidoreductase floxed (Por fl / fl), I12rg- / -, Fah- / -) mice, generated as a side product of PIRF (P450 oxidoreductase floxed (Por fl / fl), I12rg- / -, Rag2- / -, Fah- / -) strain crossing (described in Barzi et al. Nat Common 2017, 8, 39) were mated with female or male, respectively, Prkdc-SCID mice (Bosma et al. Nature 301, 527-530 (1983)) to generate the PIFS (Por fl / fl, I12rg- / -,Fah- / -, Prkdc-SCID + / + and wildtype for the Rag2 (Rag2+ / +)) mouse. The IFSA (I12rg- / -,Fah- / -, Prkdc-SCID + / +, Aavr - / - and wildtype for the Rag2 (Rag2+ / +)) mouse was then cross-bread with the PIFS (Por fl / fl, I12rg- / -,Fah- / -, Prkdc- SCID + / + and wildtype for the Rag2 (Rag2+ / +)) mouse to generate the PIFSA (POT FL / FL, II2rg- / -,Fah- / -, Prkdc-SCID + / + Aavr - / - and wildtype for the Rag2 (Rag2+ / +) mouse.

[0063] Genotyping tests for each gene were also setup in Transnetyx.com, a genotyping platform (Transnetyx Incorporation, Memphis, TN, USA).Human hepatocyte isolation

[0064] Human hepatocytes were isolated by the two-step collagenase perfusion method of Berry and Friend (77?e Journal of cell biology 43, 506-520 (1969)) with the modification of Segelen (Methods in Cell Biology 13, 29-83 (1976)). In brief, the largest portal veins were cannulated with a silicon tubing system connected to a peristaltic pump, then the Ever wasflushed with ice-cold basic perfusion solution (BPS: 10 mM Hepes buffer) followed by perfusion with BPS containing 0.5 mM EGTA to prevent the formation of blood clots. The liver was then perfused with warm collagenase solutions (2 mg / dl collagenase) until the organ became soft. The liver was cut into small pieces (2-3 cm3) and hepatocytes were released into die solution by applying minor shear stress (with forceps) on die pieces. Hepatocytes were immediately washed in ice-cold BPS containing 0.5% BSA and centrifuged (3-times 50xg, 5 min). Viability was assessed by trypan blue exclusion. Alternatively human hepatocytes were purchased from Lonza (lonza.com) (Catalog #: HUCPG).Hepatocyte transplantation into mice and validation of human albumin ELISA in the murine serum

[0065] Hepatocytes (l-3xl0® / mouse) were transplanted into the murine liver of IPS, PIES, IFSA and PIFSA mice by splenic injections as originally described for mouse hepatocytes (Ponder,, etal., Proc Natl Acad Sci USA 88, 1217-1221 (1991)). In brief, the abdominal cavity was opened by an abdominal incision, and 1-3x10® human hepatocytes in a volume of 100 pl PBS were injected into the spleen. Immediately after transplantation. selection pressure towards transplanted human hepatocytes was applied by withdrawing the drug nitisinone (NTBC) from the drinking water in the following steps: 2 days at 25%, then 2 days at 12% and eventually 2 days at 6% of the colony maintenance dose (100% = 7.5mg / l) prior to discontinuing the drug completely (Bissig el al., Journal of clinical investigation 120, 924- 930 (2010)). Mice with clinical symptoms (hunched posture, lethargy, weight loss, etc.) were put back on 100% nitisinone for a few days before once again being weaned off the drug as described above, hi order to determine the extent of human chimerism, human albumin was measured by ELISA (Bethyl laboratories, kit # £88- 129) in the murine blood, having previously shown that human albumin levels correlate with the level of human chimerism assessed by immunostaining of human hepatocytes (Bissig et al., (2010)) The Journal of clinical investigation 120, 924-930). Human Albumin levels in murine serum were determined at a dilution of 1:100 (1-3 months after transplantation) to 1:10,000 (after 3 months of transplantation) using the manufacturer’s recommendations.Euthanasia and harvesting of chimeric livers

[0066] Humanized mice were euthanized via carbon dioxide overdose, to effect. Mice were decapitated after overdose to ensure immediate death. This method of euthanasia is consistent with the recommendations of the Panel of the American Veterinary Medical Association.

[0067] Liver were fixed in 4% paraformaldehyde (PFA) over night at 4°C and then stored at 70% ethanol until standard paraffin embedding.Immunostaining of chimeric livers

[0068] Paraffin-embedded slides were deparaffinated, rehydrated, and treated with antigen retrieval citrate buffer (pH 6.0) (for LDH) or with Tris-EDTA buffer (pH 9.0) (Abeam) antigen retrieval (for BCKDHB) for 30 min at 95°C degrees. Endogenous peroxidase was quenched using 3% hydrogen peroxidase solution (Sigma-Aldrich).

[0069] For LDH, avidin and biotin were blocked using the Avidin / Biotin kit following file manufacturer’s instructions. After blocking with serum (Vector Laboratories, Cat:PK-2200), monoclonal mouse anti-hLDH antibody (Santa Cruz, CatLDH (H-10) sc- 133123) was diluted 1:100 in antibody diluent (Abeam, Catab64211) and incubated at 4°C overnight. Samples were washed in lx PBS, and incubated with anti-mouse biotinylated secondary antibody for 30 minutes and the signal was amplified using an avidin-biotin complex (ABC) detection system (Vector Laboratories, Cat:SP-2001) according to tiie manufacturer’s instructions. For BCKDHB, protein block (Abeam, Cat:ab64226) was applied for 1 hour at room temperature before rabbit anti-BCKDHB antibody (Thermo Fisher, Cat:13685-l-AP) diluted 1:300 in antibody diluent and incubated at 4°C overnight. Slides were washed with lx PBS and incubated with HRP-labelled Polymer Anti-Rabbit (Dako, Cat:K4003) at room temperature for 30 minutes. Immunostaining was developed with a DAB (3,3’- diaminobenzidine tetrahydrochloride) substrate kit (Abeam, Cat:ab64238). Counterstaining was performed using hematoxylin solution (Richard- Allan Scientific) and bluing solution (Richard-Allan Scientific). Cytoseal (Epredia) was used for mounting slides.Measurements of Engraftment

[0070] Three or four months after transplantation mice were bleed by puncture of the venous eye plexus. Plasma was diluted 1:10,000 and evaluated for human albumin levels using ELISA (hALB ELISA, Bethyl laboratories).Statistics

[0071] Sample sizes for experiments were determined by estimated differences between groups and availability of humanized mice. No randomization of animals before allocation to experimental groups nor blinding of experimental groups was deme. Statistical analysis was performed using PRISM version 6.0 software (Graph Pad software) using Mann- Whitney test Statistical significance was assumed with a p-value <0.05 (*), 0.01(**) and 0.001(***). Bars in graphs represent mean ±SEM unless noted otherwise. Group size (N) represents biological sample size.Results

[0072] The levels of human-specific albumin increased significantly in file livers humanized FIS mice, indicating a large proportion of human liver cells were present in these mice. (FIG. l).s

[0073] Successfill engraftment of three independent donor hepatocytes into IFS (H2rg- / - / Fah- MPrkdc-SCID + / +) mice was achieved four months after transplantation (FIG. 2). Each dot in FIG.2 represents one transplanted and evaluated mouse sample (N=4 for donor A and B and N=7 for donor C).

[0074] The levels of humanization in the IFS mouse was significantly greater than that achieved in file FRG mouse three months after transplantation (FIG. 3).

[0075] Immunostaining of chimeric livers of IFS mice three months after transplantation showed a great degree of chimerism in the liver. FIG. 4 shows a representative picture, indicating that human hepatocytes are engrafting over the whole functional liver acinus (zone 1, 2 and 3).

[0076] Similarly, Immunostaining showed large areas of human liver chimerism in PIFS (For fl / fl, I12rg- / -,Fah- / -, Prkdc-SCID + / + ) mice with deleted P450 oxidoreductase (For) gene (FIGs. 5A-5C), in IFSA (I12rg- / -,Fah- / -, Prkdc-SCID + / + AAVR" / ") mice (FIG. 6) and in IFS (I12rg- / -,Fah- / -, Prkdc-SCID + / + ) mice (FIG. 7).

[0077] Chimerism in IFSA mice was greater than in TTRFA (transgene-fiee, I12rg- / - / Rag2- / - / Fah- / -) control mice (Barzi etal, Nature communications 8, 39 (2017)), as assessed by human albumin levels in the murine plasma 3 months after transplantation (FIG. 8).

Claims

CLAIMSWhat is claimed is:

1. A genetically modified non-human animal, comprising a deletion of or mutation in the H2rg gene, the Fah gene and fire Prkdc gene.

2. The genetically modified non-human animal of claim 1, further comprising a deletion of or mutation in the aavr gene.Hie genetically modified non-human animal of claim 1 or 2, further comprising a deletion of or mutation in the por gene.

4. The genetically modified non-human animal of any one of claims 1-3, further comprising a deletion of or mutation in the tyrosinase gene.

5. The genetically modified non-human animal of any one of claims 1-4, wherein the non-human animal is a primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig.

6. The genetically modified non-human animal of any one of claims 1-4 wherein the nonhuman animal is a rodent.

7. The genetically modified non-human animal of any one of claims 1-4, wherein the non-human animal is a mouse.

8. The genetically modified non-human animal of any one of claims 1-4, wherein the non-human animal is a rat.

9. The genetically modified non-human annual of any one of claims 1-8, wherein at least 75% of the hepatocytes present in the non-human animal are human hepatocytes.

10. The genetically modified non-human animal of any one of claims 1-8, wherein at least 80% of the hepatocytes present in the non-human animal are human hepatocytes.

11. The genetically modified non-human animal of any one of claims 1-8, wherein at least 85% of tiie hepatocytes present in the non-human animal are human hepatocytes.

12. The genetically modified non-human animal of any one of claims 1-11, wherein the non-human animal comprises a wildtype rag2 gene.

13. The genetically modified non-human animal of any one of claims 1-12, wherein die non- human animal is immunodeficient.

14. A method of evaluating hepatotoxicity of a test substance, the method comprising(a) administering a test substance to the genetically modified non-human animal of any one of claims 1-13;(b) measuring at least one indicator of toxicity in the genetically modified non- human animal to which the test substance is administered; and (c) evaluating the effect of die test substance on human hepatocytes by comparing the indicator of toxicity measured in the genetically modified non-human animal to the same indicator of toxicity measured in a genetically modified non- human animal to which no test substance is administered.

15. The method of claim 14, wherein the test substance is a medicament intended for human use.

16. The method of claim 14 or 15, wherein the at least one indicator of toxicity is blood human albumin, body weight, liver-weight-to-body- weight ratio, total blood albumin, total blood protein, blood ALT, blood AST, or total blood bilirubin17. A method for preparing a chimeric non-human animal comprising human hepatocytes, comprising steps of: a. providing a non-human animal comprising a deletion of or mutation in file Il2rg gene, the Fah gene and the Prkdc gene; and. b. transplanting human hepatocytes into the non-human animal.

18. The method of claim 17, further comprising a deletion of or mutation in the aavr gene, the por gene, and / or the tyrosinase gene.

19. The method of claim 17 or 18, wherein the non-human animal is a primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or pig.

20. The method of any one of claims 17-19 wherein the non-human animal is a rodent.

21. The method of any one of claims 17-19, wherein the non-human animal is a mouse or rat.

22. The method of any one of claims 17-21, wherein at least 75% of the hepatocytes present in the non-human animal are human hepatocytes.

23. The method of any one of claims 17-21, wherein at least 80% of the hepatocytes present in the non-human animal are human hepatocytes.

24. The method of any one of claims 17-21 , wherein at least 85% of the hepatocytes present in tire non-human animal are human hepatocytes.

25. The method of any one of claims 17-24, wherein the non-human animal lacks autogenous hepatocytes.

26. The method of any one of claims 1-25, wherein the non-human animal comprises a wildtype rag2 gene.

27. The method of any one of claims 1-26, wherein the non-human animal is immunodeficient.

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